New antimicrobials are urgently needed to address the emergence of drug resistance in Gram-negative pathogens. Apramycin is an aminocyclitol aminoglycoside that has impressive activity spectrum against carbapenem-resistant Enterobacteriaceae (CRE), Acinetobacter baumannii, Pseudomonas aeruginosa, and Staphylococcus aureus. In contrast to aminoglycosides such as plazomicin, it retains activity against strains expressing widely circulating ribosomal methylases that are found at high frequency in NDM-1 carbapenemase strains. Importantly, apramycin also does not appear to have significant nephrotoxicity and ototoxicity side effects that have limited clinical use of other aminoglycosides. We have demonstrated that apramycin is rapidly bactericidal in vitro and in vivo in an A. baumannii murine thigh infection model. Importantly, there is only a single known aminoglycoside modification enzyme (AME) in Gram-negatives, present in ~30% of CRE strains, (extremely rare to absent in A. baumannii and P. aeruginosa), that inactivates apramycin through acetylation at the C-3 amine position in the 2-deoxystreptamine (2-DOS) ring. Based on the underlying compelling properties of apramycin, we propose to address the targeted hypothesis that combinatorial modification of 2-DOS will block AME modification, thereby expanding activity spectrum, and increase potency, defined by lowering of the minimal inhibitory concentrations against target Gram-negative pathogens and/or enhanced in vivo antimicrobial exposure defined by preliminary snapshot pharmacokinetic indices. This exploration will be accomplished in one specific aim, which will combine medicinal chemistry approaches with functional assays to assess activity spectrum, potency, selectivity, and metabolism. In the past, medicinal chemistry evaluation of the apramycin scaffold made use of semi-synthetic approaches based on modification of the existing apramycin natural product. These approaches necessarily placed significant constraint on chemical space available for exploration. The innovation of this proposal relies on use of a de novo total synthesis and novel glyco-medicinal chemistry approaches to substitute 2-DOS variants into the apramycin scaffold. The near-term goal of this two-year, exploratory R03 pilot proposal is to address potential of total synthetic glyco-medchem. approaches to identify tractable, potent analogues worthy of further exploration. The long-term goal is to develop apramycin analogues that have compelling properties as therapeutics against multidrug-resistant pathogens.